Adaptation of Hollow-Fiber-Based Cell Culture Technology for the Manufacturing of (1) Neo-Islets, Employed for the Treatment of Type 1 and Type 2 Diabetes Mellitus, and (2) the Generation of Exosomes from Various Cell Types, Used in the Treatment of Different Organ Injuries and Diseases

ABSTRACT

Disclosed herein is a method of generating exosomes (extracellular nanovesicles) and/or neo-islets from mesenchymal or adipose stem cells or islets, or other cells in a Hollow-Fiber-based Cell Expansion (HFCE) System. Such exosomes and/or neo-islets may be used for the treatment of T1DM, T2DM, or associated microvascular disease.

FIELD

This disclosure relates generally to the fields of cell biology andmedicine. In particular this disclosure relates to the production ofcells and exosomes for use in treating subjects and more particularly tothe production of cells and exosomes that are useful in treatingDiabetes mellitus

BACKGROUND

Mesenchymal Stem or Stromal Cells (MSC) from bone marrow, fat, umbilicalcord, placenta, amniotic fluid, and other sources are non-embryonic,“adult” stem cells that possess potent anti-inflammatory,anti-apoptotic, mitogenic, angiogenic and vasculo-protective, immunemodulating, anti-fibrotic, anti-thrombotic and anti-biotic paracrineactivities that have been used for the promising treatment of variousacute and chronic diseases and organ injuries. Most specifically, wehave shown (Westenfelder C, Gooch A. et al. STEM CELLS TranslationalMedicine Volume 6, Issue 7, pages 1631-1643, JULY 2017) that theco-aggregation of allogeneic MSCs and allogeneic, culture expandedpancreatic islet cells durably corrects T1DM in a mouse model ofauto-immune T1DM that closely resembles human and canine T1DM (NODmice). The MSC component of NIs provides robust protection against allo-and auto-immune attacks of insulin-producing endocrine islet cells invivo, making the use of potentially toxic anti-rejection drugsunnecessary, as these can cause serious infections, malignancies, damageof transplanted islets in the liver and kidney disease. In addition, theNI technology allows the generation of 80+ therapeutic doses from onepancreas donor, which is in striking contrast to pancreatic islettransplantation protocols, where a diabetic recipient needs 4 or moredonors for one single treatment. MSCs in culture release beneficialcytokines and growth factors that mediate their pleiotropic effects. Inaddition, MSCs, like essentially all cells, release

Exosomes (30-100 nm in diameter) into their microenvironment that aretaken up by adjacent cells, which, in turn, results in paracrinesignaling in the target cells. Signaling occurs by the lateral transferof mRNAs, miRNAs, proteins, and lipids, which together are known toexert beneficial effects that are essentially identical to those ofintact MSCs.

BRIEF SUMMARY

Described herein are a) mesenchymal and/or adipose stem cells; b)pancreatic islet cells; c) mesenchymal and/or adipose stem cell-derivedexosomes; and d) exosomes derived from other cell types such asendothelial, muscle and other cultured cells.

Further described are Neo-Islets (NIs), where NIs comprise mesenchymaland/or adipose stem cells; and pancreatic islet cells.

Disclosed herein is a method of generating exosomes (extracellularnanovesicles). Such methods may include the expansion of mesenchymal oradipose stem cells or islets, or other cells in a Hollow-Fiber-basedCell Expansion (HFCE) System and collecting exosomes released by theculture expanded mesenchyma, adipose stem cells, or other cells from theHFCE System.

In embodiments of the method, the cells are expanded to 70-95%confluency. In further embodiments, the culture medium perfused throughthe HFCE system may be adjusted to enhance the release of exosomes intothe lumens of the hollow fibers.

Embodiments include exosomes produced by such methods as well as methodsof treating subjects suffering from T1DM, T2DM, or associatedmicrovascular disease by treating the subject with the exosomes producedby the above methods. In particular embodiments the exosomes have beencryopreserved for at least two years prior to treatment.

Provided are embodiments of a method for generating Neo Islets.Particular embodiments include culture expanding mesenchymal or adiposestem cells and pancreatic islet cells in HFCE System and collecting NeoIslets from the HFCE System. In embodiments of the method, the cells areexpanded to 70-95% confluency.

In embodiments the Neo Istels may comprise a) dedifferentiated isletcells and mesenchymal or adipose stem cells and/or redifferentiatedislet cells and mesenchymal or adipose stem cells. In particularembodiments wherein the islet cells and stem cells are present in theNeo Islets at an islet cell:stem cell ratio of about 1:100, 1:75, 1:50,1:25, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 25:1, 50:1, 75:1, or 100:1.

In certain embodiments the collected Neo Islets are placed in a hydrogeland/or are encapsulated.

Embodiments include neo-islets produced by such methods.

Embodiments include methods of treating a subject suffering from T1DM orT2DM, and associated microvascular disease with neo-islets produced bythe above described methods.

Also described are methods of treating a subject with T1DM or T2DM, themethods comprising: administering to the subject NIs with or withoutexosomes derived from MSCs or other cultured cells.

The here disclosed NI technology harnesses the pleiotropic functions ofMSCs, where appropriate, and of their Exosomes where needed in order torepair and protect the affected capillary system in the diseases anddisorders listed above.

“Treating” or “treatment” does not require a complete cure. It meansthat the symptoms of the underlying disease are at least reduced, and/orthat one or more of the underlying cellular, physiological, orbiochemical causes or mechanisms causing the symptoms are reduced and/oreliminated. It is understood that reduced, as used in this context,means relative to the state of the disease, including the molecularstate of the disease, not just the physiological state of the disease.

As used herein “therapeutically effective amount” is an amountsufficient to act as a treatment as defined above. This may bedetermined by, e.g., standard techniques used to monitor and/or diagnosea particular disease state.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps, but also includes the more restrictive terms “consisting of” and“consisting essentially of.

In conclusion, the purpose of using a customized Hollow-Fiber-based CellCulture system is to allow the scaled-up and highly efficient productionof MSCs, islet cells and thus NIs for the treatment of diabetes. Inaddition, various cultured cell types in a Hollow-Fiber-based CellCulture system can be programmed to generate large numbers of Exosomesin which therapeutically desired and unique therapeutic qualities arepotentiated and used for the treatment of specific diseases inPersonalized and Regenerative Medicine. It is also envisioned that thebroad scale use of the developed, high efficiency Hollow-Fiber-basedCell Culture Technology will prove cost saving for the entire HealthCare System.

DETAILED DESCRIPTION

The illustrations presented in this disclosure are not meant to beactual views of any particular compositions, but are merelyrepresentations employed to describe illustrative embodiments. Thus, thefigures are not necessarily to scale.

In this disclosure, the term Mesenchymal Stem Cell means and includesmesenchymal and/or adipose stem cells; and b) mesenchymal and/or adiposestem cell-derived exosomes.

While certain illustrative embodiments have been described in connectionwith the figures, those of ordinary skill in the art will recognize andappreciate that the scope of this disclosure is not limited to thoseembodiments explicitly shown and described in this disclosure. Rather,many additions, deletions, and modifications to the embodimentsdescribed in this disclosure may be made to produce embodiments withinthe scope of this disclosure, such as those specifically claimed,including legal equivalents. In addition, features from one disclosedembodiment may be combined with features of another disclosed embodimentwhile still being within the scope of this disclosure, as contemplatedherein.

This disclosure relates generally to the manufacturing of Neo-Islets forthe treatment of Type 1 Diabetes mellitus in companion animals such asdogs and in human subjects. Neo-Islets (NIs) are co-aggregates ofculture expanded pancreatic islet cells and cultured Mesenchymal StemCells (MSCs) obtained from bone marrow or adipose tissue. TheHollow-Fiber-based Cell Culture technology is a highly efficient,enclosed system, therefore not requiring a clean room that is currentlyused for the expansion of attached MSCs and other cells both forresearch and clinical use and for suspension culture of various types ofblood cells.

Therapeutic targets for the here disclosed technology include both Type1 and Type 2 Diabetes mellitus and their multiorgan complications(kidneys, eyes, heart, neurological and others), vasculitides,auto-immune diseases, sepsis, acute and chronic kidney diseases,cardiovascular and neurological diseases, solid organ transplantation,vascular rejection, wound healing, atherosclerosis, aging, and variousdegenerative retinal and neurological diseases. More specifically,disclosed embodiments relate to the therapeutic use of Mesenchymal StemCells from various sources as a critical component of NIs and their

Exosomes, to repair and protect and stabilize the diseasedmicrovasculature and thereby improve outcomes and survival both incanines and humans with T1DM.

It is envisioned that a Hollow-Fiber-based Cell Culture (HFCE) systemcan be used, with modifications, for the expansion of islet cells, theformation of therapeutic doses of NIs, and the generation of Exosomesfrom cultured MSCs and other cells that are programmed to impartspecific therapeutic characteristics on released Exosomes for thetreatment of various diseases, such as acute renal failure, chronickidney disease, strokes, heart attacks, various auto-immune diseases,organ transplantation, and others

The use of an HFCE system to produce exosomes and NIs: (See also aboveparagraph). Such HFCE systems are in some aspects similar to theconfiguration of clinically used hemodialysis cartridges. These systemsahve the distinct advantage of providing a highly favorable surface areato culture medium ratio and flexibility regarding changes in cultureconditions of both attached cells (e.g. Mesenchymal Stem, islet, andother cell types) and cells in suspension culture, a technique that isused for the manufacturing of Neo-Islets. In addition, various attachedcell types that are expanded in a HFCE system can be programmed torelease exosomes with optimized therapeutic characteristics such asimmune-isolation, anti-inflammatory, cytoprotective, angiogenic, andothers. Together, the efficacy and therefore costs of cell culturing,Neo-Islet and exosomes production in a suitable HFCE system, areexpected to be substantially more favorable than in conventional culturesystems.

Production of Exosomes (extracellular nanovesicles) are spontaneouslyreleased from MSC, ASC and most other cell types when efficientlycultured in a suitable HFCE System (a bioreactor) or flask system. Theycan be continuously harvested, characterized (surface marker expression,miRNA, RNA, DNA, peptides, cytokines and lipid cargo), cryopreserved orfreshly used. The utilized cell culture media in the HFCE System arestandard media that are used by all experts in the field. However,modifications of the culture media can be employed to generate exosomesthat possess desirable, therapeutic characteristics, such as augmentedanti-inflammatory, immune-modulating and trophic effects(anti-apoptotic, mitogenic, angiogenic and others).

The production of NIs with a HFCE System, as described above, utilizesculture expanded pancreatic islet cells (insulin, glucagon,somatostatin, PPY producing cells and other non-endocrine cells) andculture expanded MSCs or ASCs. The suspension co-culture of islet cellsand MSCs or ASCs in a suitably modified HFCE System results in theefficient generation of NIs that, after appropriate characterization(relevant gene expression profiles, glucose-sensitive insulin releasetest, etc.) can be used fresh for the treatment of

T1DM or they can be cryopreserved for later use.

Regarding the treatment of subjects using NIs, the contents of U.S.application Ser. No. 15/261,750 are incorporated herein in its entiretyby this reference.

EXAMPLES

The following examples are provided for illustration purposes only andare not to be construed as limiting the disclosure to the embodimentsspecifically disclosed therein.

Both type 1 and type 2 diabetes mellitus progressively result inwide-spread end organ damage, affecting the retina, coronaries, thenervous system, kidneys and other organs though microvascular diseasethat injures the capillary beds in these organs through endothelial andpericytes dysfunction, microvascular obstruction and inflammation,vasoconstriction, vascular leakage, coagulopathy, and fibrosis. Thismicrovascular damage also affects the capillaries of the islets ofLangerhans in the pancreas. This pathomechanisms, we hypothesized couldaccelerate the destruction of insulin producing cells and thereby hastenthe complete loss of intrinsic insulin production, i.e., greatlyaggravate the diabetic state in a patient. Accordingly, we testedwhether the parenteral administration of mesenchymal and/oradipose-derived exosomes to db/db mice with advanced diabetic diseasewould improve glycemic control. This was observed, together withimprovement in diabetic kidney disease.

In analogy to the microvascular disease of diabetes, which shares mostpathologic features of various vasculitides, auto-immune diseases,sepsis, acute and chronic kidney diseases, cardiovascular andneurological diseases, solid organ transplantation, vascular rejection,wound healing, atherosclerosis, aging, and various degenerative retinaldiseases, we expect that the treatment with Mesenchymal and/or AdiposeStem Cells alone or their exosomes alone or in combination with theirparent stem cells will favorably affect outcomes in this large group ofmicrovascular diseases.

A type 2 diabetic male subject presents with chronic kidney disease.MSCs are isolated from the subject and expanded in culture. The expandedMSCs are administered to the subject in an amount sufficient to improvethe symptoms from which the subject is suffering that are related tomicrovasculature issues.

Detailed examples of using an HFCE System to produce exosomes and NIs:

It is envisioned that the HFCE System is used to produce exosomes fromcultured MSCs, ASCs, and other cells of interest as shown in FIG. 1. Thecells of interest from which exosomes (MSCs or ASCs, other cells) or NIs(islet cells, MSCs or ASCs) are produced will be loaded, suspended insuitable culture media, into the top port (FIG. 1). Defined culturemedia will perfuse the internal and external spaces of the hollow fibersthat contain cells to be expanded or co-cultured (MSCs or ACSs withislet cells). Once the desired degree of cell expansion within the HFCESystem is reached, cells are detached and harvested, at the bottom ofthe HFCE System. Once the desired degree of cell clustering is reached,NIs are harvested at the bottom of the HFCE System. Exosomes fromcultured cells are collected without detaching the cultured cells.

The treatment of diabetic (Type 1) subjects with the NIs consists ofadministering a body weight-based dose of this composition into theintraperitoneal space of such a subject, using ultrasound guidance andlocal anesthesia. The Exosome-based treatment of subjects with variousdiseases (autoimmune, transplantation-related complications, ischemicinjury, vasculitis, liver disease, neurodegenerative, retinal, cardiac,renal and other disorders) consists on the intravenous administration ofa predetermined dose of functionally “customized” (see above) Exosomes.

What is claimed is:
 1. A method of generating Exosomes (extracellularnanovesicles), the method comprising: expanding mesenchymal or adiposestem cells or islets or other cells in a Hollow-Fiber-based CellExpansion (HFCE) System; and collecting Exosomes released by cultureexpanded mesenchymal or adipose stem cells or other cells from the HFCESystem.
 2. The method according to claim 1 wherein the cells areexpanded to 70-95% confluency.
 3. The method according to claim 1further comprising adjusting culture medium perfused through the HFCEsystem to enhance the release of exosomes into the lumens of the hollowfibers.
 4. Exosomes produced by the method of claim
 1. 5. A method oftreating a subject suffering from T1DM or T2DM, and associatedmicrovascular disease, the method comprising: treating the subject withexosomes produced by the method of claim
 1. 6. The method according toclaim 5, wherein the exosomes have been cryopreserved for at least twoyears prior to treatment.
 7. A method for generating Neo Islets, themethod comprising: culture expanding mesenchymal or adipose stem cellsand pancreatic islet cells in a Hollow-Fiber-based Cell Expansion (HFCE)System; and collecting Neo Islets from the HFCE System.
 8. The methodaccording to claim 7 wherein the cells are expanded to 70-95%confluency.
 9. The method according to claim 7, wherein the Neo Istelscomprise: a) dedifferentiated islet cells and mesenchymal or adiposestem cells; or b) redifferentiated islet cells and mesenchymal oradipose stem cells.
 10. The method according to claim 9, wherein theislet cells and stem cells are present in the Neo Islets at an isletcell:stem cell ratio of about 1:100, 1:75, 1:50, 1:25, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 25:1, 50:1, 75:1, or 100:1.
 11. The method according to claim7, wherein the collected Neo Islets are placed in a hydrogel.
 12. Themethod according to claim 7, wherein the collected Neo Islets areencapsulated.
 13. A method of treating a subject suffering from T1DM orT2DM, and associated microvascular disease, the method comprising:treating the subject with neo-islets produced by the method of claim 7.14. Neo-Islets produced by the method of claim 7.